Fermentation is a process to produce one or more products from one or more substrates through use of a biocatalyst, wherein the biocatalyst can be a whole microorganism, an isolated enzyme, or any combination thereof.
In a batch fermentation, fermentation begins with a culturing process in which the medium is inoculated with the desired microbial organism. Growth or metabolic activity then occurs. The metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Typically, the growth rate of the microbial cells proceeds through a static lag phase, to a high-growth log phase (or exponential growth), and finally to a stationary phase, wherein growth is diminished or halted. Although production of the microbial product typically occurs during the high-growth log phase, this phase of growth cannot continue indefinitely because the medium becomes depleted of nutrients and enriched with products, if the product is secreted, and byproducts, as a result of the cultured organisms' growth. Byproducts may comprise among other things, polysaccharides, carbohydrates, amino acids, proteins, salts and various organic acids such as lactic acid, acetic acid, formic acid, proprionic acid, pyruvate, fumarate, citrate, isocitrate, glyocylate, succinate, α-ketoglutarate and malonates.
Fermentation is an important technology for biosynthesis of a variety of microbial products, including amino acids, ethanol, polyunsaturated fatty acids and antibiotics. The fermentative production and commercialization of a few chemicals have been reported (W. Crueger and A. Crueger, Biotechnology: A Textbook of Industrial Microbiology, Sinauer Associates: Sunderland, Mass., pp 124-174 (1990); B. Atkinson and F. Mavituna, Biochemical Engineering and Biotechnology Handbook, 2nd ed.; Stockton: N.Y., pp 243-364 (1991)). Biocatalytic processes, however, frequently suffer from several well-known limitations which may include: 1) a relatively small range of products; 2) low yields, titers and productivities; 3) difficulty recovering and purifying products from aqueous solutions; and, 4) generation of unwanted byproducts. Integrating upstream metabolic engineering (i.e., product synthesis) with downstream bioprocess engineering (i.e., product separation and process design) is critical to reap significant value from industrial fermentation because process limitations increase the cost of manufacture of the product of interest.
Although various biochemical, physiological and chemical/physical factors can affect the productivity of a biocatalytic process, important factors include the efficiency of the conversion of the substrate to product and the optimization of energy/carbon flow into the biochemical pathway that results in the product of interest. In consideration of these factors, a variety of metabolic engineering techniques have been developed to facilitate up-regulating desirable biochemical pathways and down-regulating undesirable biochemical pathways, such as those that compete with the biosynthetic pathway of interest or those that interfere with production of a particular end-product.
The present disclosure concerns the accumulation of malonates as byproducts during the fermentative synthesis of a product by an organism. Specifically, high productivity and minimal waste byproduct are achieved by engineering the organism to express a heterologous malonyl-CoA synthetase to enable the reaction: malonate+ATP+CoA→malonyl-CoA+AMP+pyrophosphate [“PPi”]. Conversion of the byproduct malonate to malonyl-CoA permits synthesis of fatty acids within the organism, thereby avoiding accumulation of malonate “byproducts” that can not be further utilized during the fermentation. This thereby avoids carbon and energy waste within the organism, reduces the amount of base required to maintain an optimal pH range during the fermentation process, and reduces the amount of byproduct organic acids that require neutralization within the fermentation waste steam.
Heterologous malonyl CoA synthetases have been previously expressed in microbial organisms to enable enhanced production of various polyketides (U.S. Pat. No. 6,939,691, U.S. Pat. App. Pub. No. 2003/0073205). As summarized in Lombó F., et al. (Biotechnol. Prog., 17(4):612-7 (2001)), the productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the in vivo availability of precursors derived from α-carboxylated CoA thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. Expression of a malonyl-CoA synthetase can alleviate this limitation and significantly increase polyketide production. Previous disclosures do not contemplate expression of a heterologous malonyl CoA synthetase to reduce production of malonates and thereby avoid carbon and energy waste by the organism.
Applicants have solved the stated problem whereby malonate “byproducts” accumulate during the fermentation of an organism, leading to carbon and energy waste, reduced synthesis of the product of interest and production of waste streams that require neutralization (thereby increasing the overall cost of manufacture). Novel organisms expressing heterologous malonyl CoA synthetase proteins are described herein.